Vernix caseosa (vernix) is a lipid-rich naturally occurring skin protectant composed of sebum, epidermal lipids, and desquamated epithelial cells. It covers the skin of the developing fetus in utero while the fetus is completely surrounded by amniotic fluid. Vernix consists of hydrated cells dispersed in a lipid matrix. Natural vernix comprises about a 10% lipid fraction by weight, about a 10% protein fraction by weight, and about an 80% volatile fraction by weight. The lipid matrix undergoes a transition to a more fluid form at physiological temperatures and with the application of shear forces, such as those encountered with movement. Vernix is a covering for the skin of the fetus that resembles the stratum corneum except that it lacks multiple rigid desmosomal connections. Consequently, vernix exhibits a viscous fluid character.
The lipid component of vernix has been reported in J. Invest. Dermatol. 78:291(1982); Lipids 6:901(1972); J. Clin. & Lab. Investigation 13:70 (1961); J. Invest. Dermatol., 44:333 (1965); and U.S. Pat. No. 5,631,012, each of which is incorporated by reference herein in its entirety. Lipids, defined herein as fats or fat-like substances, include lecithin and other phospholipids, squalene, waxes, wax esters, sterol esters, diol esters, triglycerides, ceramides in which the fatty acid components may be one or more of the following: α-hydroxy 6-hydroxy-4-sphingenine, α-hydroxy phytosphingosine, α-hydroxy sphingosine, ester linked ω-hydroxy 6-hydroxy-4-sphingenine, non-hydroxy phytosphingosine, non-hydroxy sphingosine, and/or ester linked ω-hydroxysphingosine; free sterols, and four classes of fatty acids ranging in chain length from C12 to C26 (straight chain saturated, chain unsaturated). The lipid fraction may contain, with the relative percentages indicated, squalene (9%), ceramides (10%) aliphatic waxes (12%), sterol esters (33%), diesters (7%), triglycerides (26%), free sterols (9%), and other lipids (4%). The fatty acids within the aliphatic waxes may be branched and the branched fatty acids may be methylated.
Because of its anticipated skin maturation and protectant properties, vernix appears to have promise as a clinically effective therapeutic agent. Application of vernix to clinical use, however, has been limited by the difficulty in obtaining samples of sufficient volume, the possibility of disease transmission, and the physical properties of native vernix.
Regarding its physical properties, vernix in utero is a tractable semi-solid, whereas vernix ex utero is a nonhomogeneous intractable compound with a consistency comparable to cheese or hardened cake frosting. Vernix is not completely soluble in conventional solvents such as absolute ethanol, 95% ethanol, 2-propanol, and combinations of chloroform and methanol. Thus, controlled and uniform administration of vernix to a surface is difficult. While it has been reported that the surfactant polysorbate 80 (Tween 80) may solubilize vernix, we previously reported that Tween 80 is toxic to living cells and therefore cannot be used clinically, i.e. for direct application to compromised skin. Tween 80 may have unwanted longer term effects in some populations. Isolated reports disclose vernix directly scraped from a newborn for smearing over wounds (U.S. Pat. No. 1,718,947A) or an artificial lipid composition as a cosmetic moisturizer (U.S. Pat. No. 5,631,012).
Natural vernix contains proteins which, in general, are multi-determinant antigens. Thus, the protein component of vernix may be capable of inducing an immune response and reacting with the products of an immune response. Proteins with a greater degree of complexity generally provoke a more vigorous immune response.
The protein fraction of natural vernix consists of epidermally derived proteins, primarily keratin and filaggrin, trace amounts (micromolar to millimolar concentrations) of regulatory proteins such as epidermal growth factor, and trace amounts (nanomolar to micromolar concentrations) of surfactant protein such as surfactant associated protein-A and surfactant associated protein-B.
Because virtually all proteins are immunogenic in an appropriate individual, and because of the different type and complexity of proteins in natural vernix, at least some immune response would be anticipated when vernix is applied to non-self (other than the baby and/or the mother). The response encompasses physical, biochemical, and molecular changes, such as stimulation of T cells, B cells, and macrophages, hypersensitivity reactions or allergic reactions, inflammation, fever, etc.
We have previously reported that synthetic vernix may be produced by mixing one part of natural vernix, removed from the infant at the time of delivery, with any of the following components in the proportions indicated: either about 0.005 to about 0.05 parts phospholipid, or trace amounts of about nanomolar to micromolar concentrations of pulmonary surfactant proteins such as surfactant A and/or surfactant B, or 5 parts dimethylsulfoxide (DMSO), or 1 part amniotic fluid, or combinations of the above. Alternatively, synthetic vernix may also be produced by combining lipids to comprise about a 10% fraction of the entire volume, proteins to comprise about a 10% fraction of the entire volume, and water to comprise the remaining about 80% of the entire volume. The following lipid components are combined in the relative percentages indicated: squalene (9%), aliphatic waxes (12%), sterol esters (33%), diesters (7%), triglycerides (26%), free sterols (9%), and other lipids (4%). The fatty acids within the waxes may be branched and the branched fatty acids may be methylated. The protein components, combined to constitute about a 10% fraction, are epidermally derived proteins, primarily keratin and filaggrin, with trace amounts of about micromolar to millimolar concentrations of regulatory proteins such as epidermal growth factor, and trace amounts of about nanomolar to micromolar concentrations of surfactant protein such as surfactant A and surfactant B.
Skin cleansing formulations generally contain surfactants that emulsify soils on the skin surface for removal with a water rinse. Surfactants may be anionic, cationic, nonionic, or zwitterionic and can be in the form of a bar, a liquid, a cream, a gel, etc. Surfactants vary markedly in their effects on the skin and differ significantly in their inherent irritancy to skin. They have been shown to vary in their effects on corneocyte swelling, disaggregation, and damage. Surfactants, as well as other topical treatments, can vary greatly in their effects on the permeability barrier of skin. For example, the effects of sodium dodecyl sulfate (SDS) and acetone on human skin in vivo (biopsy specimens) were evaluated by electron microscopy. Damage to nucleated epidermal cells and disruption of lipid extrusion were observed for skin treated with 0.5% SDS, even though the upper stratum corneum was intact. Acetone treatment resulted in disruption of epidermal lipid lamellae and loss of lamellar cohesion throughout the stratum corneum.
The amount of residual material left on the skin surface after cleansing depends upon properties of the surfactant, including its interaction with calcium and magnesium in the rinse water. The amount of surfactant used in cleansing and the extent of surfactant dilution with rinsing (i.e., volume of rinse water) can impact the residual material remaining on the skin surface. Procedures for bathing newborns frequently involve minimal rinsing to minimize the cooling effects of full body water exposure. Consequently, due to the low volume and short duration of rinsing, the level of residual surfactant on newborn skin is expected to be high.
Given the irritating and drying effects of surfactants on skin, the advisability of bathing infants with cleansing products warrants re-evaluation. It has been recommended to use mild cleansers with few ingredients to minimize irritant and allergic dermatoses in infants, and to use specialized preparations for specific dermatoses that might occur (J Eur Acad Dermatol Venereol 2001;15,12). One of the functions of bathing a newborn is to remove blood and pathogens to prevent transmission to others. A study compared the pathogen colonization rate for a group of 62 infants bathed using a mild cleanser, with the colonization rate for a group of 65 infants bathed with water alone (Birth 2001;28,161). Colonization of the skin increased over time in both groups, with no difference in type or quantity of microorganisms. These data indicate that the cleanser does not impact bacterial colonization.
Various cleansing treatments on the skin of infants aged 2 weeks to 16 months were evaluated (Dermatology 1997;195,258). Parallel treatment groups of 7-10 infants were washed one time with water alone, a synthetic liquid cleanser, a synthetic bar cleanser, or a fatty acid soap. Measures of skin pH, hydration, and surface lipid content were made before and ten minutes after washing. All four treatments increased the skin pH over its starting pH, with water alone resulting in the smallest pH increase and soap resulting in the largest pH increase. All three cleansers resulted in a significantly greater pH increase than water alone, with the fatty acid soap significantly greater than the synthetic liquid or bar cleanser. The authors indicated that the tap water used in bathing was alkaline (pH 7.8-8.2), but the extraction of water-soluble amino acids in natural moisturizing factor due to bathing is expected to give rise to a higher pH. No differences were detected in skin hydration, either as a result of bathing or the cleansing product. This finding is inconsistent with the results of another study reporting decreased hydration following bathing, but in the latter study, the time after bathing was shorter (10 min versus 15 min), the base sizes of the infants were smaller, and the age ranges differed (3-6 months versus 2 weeks-16 months) (Ped Dermatol 2002;19,473). In another study determining the effects of bathing a group of infants over a two week period with a whey-based product, decreased hydration, pH and erythema were reported, but the changes were not statistically significant (Schweiz Rundsch Med Prax 1998;87,617).
The effects of water and surfactants can be further exacerbated by pre-existing skin conditions or other environmental factors. The effects of washing with a cleanser on stratum corneum and epidermal thickness were evaluated among normal and atopic (having allergic reactions, e.g., hay fever, asthma, atopic dermatitis) subjects. Soaps decreased the number of cell layers in the atopic subjects but not the normal subjects, suggesting that atopic individuals have increased susceptibility to cleansers.
The combination of washing with surfactants and decreased environmental humidity has also been investigated. In human adults, the effects of irritant dermatitis due to repeated water and/or surfactant exposure were exacerbated at decreased absolute humidity. Animals exposed to low humidity for a short time (two days) exhibited increased epidermal proliferation following surfactant (SDS) exposure, compared to animals housed at normal or high humidity. Additionally, animals exposed to high humidity for two weeks had greater epidermal proliferation after exposure to surfactant than did animals exposed to low or normal humidity for two weeks. This suggests that the effects of water and surfactants may be greater under humidity extremes in either infants or neonates.
Skin of premature neonates has poor epidermal barrier properties and increased susceptibility to damage. These factors, coupled with the overall medical instability of premature infants, must be balanced with the need for practices, such as bathing, to reduce the risk of infection. One study investigated the effects of reduced bathing frequency (once in four days compared to once a day) on skin pathogen colonization among a group of premature infants. No differences were found in skin flora on days 2, 3, or 4 after bathing (J Obstet Gynecol Neonatal Nurs 2000;29,584). Reduced bathing frequency for premature infants has been, and continues to be, recommended as a general standard of care in the neonatal nursery (J Obstet Gynecol Neonatal Nurs 1999;28,241). The specific effects of bathing on preterm infant physiology and behavior were investigated in a group of ten subjects, with responses measured ten minutes before, during, and ten minutes after the bath. Significant increases were observed for heart rate, cardiac oxygen demand, and motor behavior, accompanied by a significant decrease in oxygen saturation.
Compared to term infants, preterm infants are more susceptible to transdermal exposures due to their immature epidermal barrier. At one time, cleansing products containing 3% hexachlorophene were regularly used for full body bathing of premature neonates, but subsequent evaluations of the neurotoxic effects indicated that vacuolar encephalopathy was related to hexachlorophene exposure. The use of hexachlorophene-containing products was therefore discontinued for this population. Alternatives were proposed through testing on the control of pathogenic bacteria, and included Lactacyd (an alkyl sulfate surfactant) and Hibitane (chlorhexidine). Subsequent evaluation of Lactacyd, however, showed that it increased transepidermal water loss (TEWL) and was inappropriate for premature infants, and chlorhexidine was found to be cytotoxic to fibroblasts and keratinocytes in culture and therefore contraindicated for preterm infants.
Thus, the use of topical products on premature infant skin, including surfactants, cleansers, antiseptics, etc., must be carefully considered. Factors which govern the relative effect on the infant include stratum corneum thickness and integrity, amount of surface residue, inherent irritancy of residual materials, and partition coefficient through the stratum corneum. While the stratum corneum of preterm infants develops rapidly after birth under the influence of the relatively dry environmental condition, the barrier function is not fully competent for several weeks after birth and is more permeable to exogenous materials. In particular, bathing involves minimal rinsing of the skin surface, and thus residual materials remain on the skin surface.
Stratum corneum, the outermost surface of the skin, continually self-cleanses through the process of desquamation. In utero, during the third trimester, vernix gradually detaches from the fetal skin under the influence of mechanical stress and pulmonary surfactant, yielding turbid amniotic fluid. At birth, residual vernix on the skin surface forms the physical interface between the newborn infant and a nonsterile environment.
Most surfactants used in commercially available cleansing products interact with and alter the epidermal barrier, as evidenced by numerous studies using in vivo systems. A consideration of the response of infant skin, versus adult skin, to topically applied treatments is useful in order to determine the potential effects of topical treatments in the preterm infant. For example, the infant has a greater surface area to body weight ratio and absorbs proportionately greater quantities than adults. Tissue distribution depends on age, and tissue affinity may vary between infants and adults, leading to different overall effects.
There is, therefore, a need to determine methods and compositions that are useful in compromised skin, such as that found in a preterm infant having immature epidermal barrier structure and function, as well as normal skin.